We propose to study mechanisms of programmed cell death (PCD). In insect metamorphosis PCD is synchronous and total. Previous studies of intersegmental muscles and labial gland of the 10 g metamorphosing Manduca sexta show that cell death can require RNA and protein synthesis. We propose to examine the physiology and molecular biology of cell death and to study conserved genes in Drosophila melanogaster. We will: a. Characterize proteins synthesized during PCD in Manduca labial gland and muscle and compare the two tissues and Drosophila salivary gland. Compare common translation products from dying labial glands and muscle. Correlate changes in morphology of nuclei, DNA loss, and protein synthesis. Microsequence proteins of interest, and make synthetic peptides. Localize these proteins in dying and non-dying cells by immunocytochemistry. b. Isolate and characterize common cDNAs activated during involution of Manduca labial gland and muscle. Establish cDNA libraries from muscle and labial gland at comparable stages of involution. Isolate cDNA common to both tissues by subtractive hybridization of amplified PCR cDNA libraries. Characterize specific clones by: sequencing and comparison to known sequences; comparison of expression pattern during involution; localization of expression by in situ hybridization. c. Characterize PCD genes common to Manduca and Drosophila to assess function and mechanisms of action. Characterize temporal and tissue- specific expression of clones during Drosophila development. Southern- screen Drosophila libraries with isolated Manduca cDNA. Alter expression with P-element transformation. Mechanisms of cell death are likely conserved or use the same vulnerabilities. Our understanding should suggest new directions in therapeutics of aging, malignancy, and restoration of function.